W. M. Keck Ecological and Evolutionary Genetics Facility
The Keck Facility at the Josephine Bay Paul Center (JBPC) is equipped for high-throughput DNA template production, capillary sequencing, and “Next-Gen” sequencing. While the JBPC has priority use of the facility, it is available to certain other “outside users” within the Woods Hole community. Please be aware that the JBPC does not operate a commercial sequencing service and is only responsible for the operation of our specialized instrumentation. Users are expected to complete the majority of sample preparation and to recognize the concept of shared risk.
Next-Generation Sequencing Projects
The BPC has retired the Roche GS-FLX ’454′ and Ion Torrent PGM sequencers as of 2013. We have transitioned to Illumina platforms for amplicon tag sequencing and other applications. Our instrumentation and expertise can be used for collaborative projects and arrangement should be made with the current BPC director, Mitch Sogin. Please do not contact us about ‘fee for service’ work. See additional requirements for all Illumina sequencing here.
Next-Gen Project submission (only for existing 454 collaborations)
Template Preparation and Sanger Sequencing
Scheduling is at the discretion of JBPC sequencing personnel and will depend on demand. With this in mind, please help us keep things running smoothly by familiarizing yourself with the procedures outlined in this site. Please do not make sequencing plans until you have contacted JBPC sequencing team at: firstname.lastname@example.org.
Forms for services:
The JBPC prepares plasmid templates with a BiomekFX and a modified alkaline lysis protocol. It operates a single Applied Biosystems (AB) 3730XL capillary sequencer. This instrumentation is highly specialized and is operated only by trained personnel. JBPC also has an Agilent Bioanalyzer, Covaris S22, Sage PippinPrep, thermocyclers, and centrifuges which may be available to WH community users by arrangement. We do not provide or sell reagents for any of these, training will be required, and users will be charged on a prorated basis for any necessary repairs.
Please contact Hilary Morrison for information about costs and methods of payment.
All sequencing is done in 96- or 384-well plate format. You may run a partially empty plate, but both the BiomekFX and the 3730XL consume the same reagents regardless of whether the plate is full or not. Therefore there is no discount for a partial plate. Unfortunately combining different types of samples on the 3730XL can be problematic. Be aware that all 96 samples pass through the field of the laser and detector at the same time, and varying signal intensities can lead to problems.
Template preparation supplies for the BiomekFX are available only in large quantities. In order to avoid creating a purchasing headache for every user, JBPC provides most of the necessary supplies (see protocol) and they are included in the cost. You may supply your own, but due to the complexity of the MBL’s cost center system, no discount will be given.
Sequencing supplies for the 3730XL are more easily obtained by the individual user. The most expensive reagent, ABI’s Big Dye Terminator (BDT), may be purchased through JBPC.
You should use the cleanest possible template. Template from the BiomekFX generally works very well for sequencing, and the cost is low. Other acceptable methods of template production are available in kit form from Qiagen, Eppendorf, Millipore, and others. JBPC has a manifold available for the Eppendorf PerfectPrep kit. Contact ABI tech support if you are not certain that your kit will produce sequencing-grade template.
The “largest” reaction used by anyone in the JBPC is around 1/8X, which means a reaction that contains 1/8 of the amount of BDT in the official ABI reaction (1X). The reaction recipes are not simply scaled down in volume. Other modifications have been made. JBPC and most outside users have had good results sequencing clean plasmid templates with these lower reaction volumes. Using smaller reactions saves a substantial amount of money, but is not endorsed by ABI. Also, please remember that what works on one set of templates may not work well on another.
If you need more information about reaction set-up, Applied Biosystems has a good web site (www.appliedbiosystems.com). Particularly useful is the Applied Biosystems 3730/3730XL DNA Analyzers: Sequencing Chemistry Guide: Rev B (part number 4331467B). You can download the guides or buy a hard copy for about $5.00. If you need further help with template concentration, clean-ups, PCR sequencing, sequencing difficult regions, high G-C regions, etc. try ABI tech support. JBPC has a protocol for sequencing PCR products, which can be emailed to you.
Post-reaction clean up and resuspension
Post-reaction clean up is necessary to remove unincorporated fluorescent terminators and other unwanted reagents. Isopropanol precipitations are the preferred choice for simplicity and affordability. More information (and other methods) can be found in the Applied Biosystems 3730/3730XL DNA Analyzers: Sequencing Chemistry Guide (see above).
Cyclesequencing reaction products are fragile, and reactions should be cleaned up by precipitation within 24 hours. After precipitation they should be delivered to JBPC within 3 days. If long reads are a priority, be sure to contact JBPC to schedule your sequencing before you set up reactions.
Name your samples
This is a critical and often overlooked step. The 3730 software has specific requirements for sample names. If your sample names do not follow the guidelines below, we will not run your plate until they are repaired. This is for your own sake. Renaming after a run is possible, but highly time consuming.
Your names should be a simple list of 96 names in a single column in text format.
Do not use spaces or funny characters -@#$?+%^&*)( etc. If any of these characters are used the Data Collection software will not be able to import the sample names.
You can use a period . or an underscore _ to separate information.
The 3730 loads samples in rows of 8 (A1-H1). Your sample names layout should move down, then over. Example A1-H1, A2-H2, A3- H3, etc.
Excel is the easiest way to prepare your sample name list. Save the file in *.txt. (tab delimited text) format.
A handy Excel file for naming samples with simple consecutive numbers can be downloaded here.
Label your plate with your name and plateID. Please write your label on the long sides of the plate, as the short sides will be obscured by plate carriers during sequencing.
The best way to send us your sample names is to attach the TAB-DELIMITED TEXT file to an email to email@example.com. Samples will not be processed without a cost center and default sample names will be used if a sample name file (PLT file) is not provided. Be sure to send it as an ATTACHMENT, not as text within the body of the email.
To bring to JBPC
Your sample plate(s). Plates should be sealed with foil and kept cold and dark.
The JBPC sequencing facility is located in rooms 330/331 on the 3rd floor of the Lillie Building. The Lillie building is located on the corner of Water St and MBL St. The building is locked after hours and on weekends. JBPC sequencing personnel are usually available during regular business hours. You may leave your labeled plate(s) in the freezer. The correct freezer is the one just inside room 330. Samples should be placed on 4th shelf down from the top. If you are new to the process, please check in with a member of the sequencing staff (Leslie or Bette).
Retrieving your data
Shortly after your sequences are finished, you should receive an email from JBPC with a quality assessment and link to the download site. If you are a first time user, we will need to know your lab affiliation to add your email address to the account and to give you a password.
To download a zip from the Sequencing downloads site directly to your JBPC bioinformatics user account without having to transfer to your local computer first, you can use the command line utility ‘curl’:
Here are some examples:
(Note: Unless your sequencing username is jdoe , you will probably need to substitute jdoe in the username and the URL for whatever your sequencing facility username is.)
curl -s -k -O -u jdoe https://sequencing.mbl.edu/downloads/users/jdoe/150207_EDC1.zip
Logs into the JBPC sequencing site at the URL specified as user ‘jdoe’ and downloads a file named 150207_EDC1.zip and saves it into a file of the same name in your current working directory. If the file already exists in the current working directory, it will be overwritten.
Note: curl will prompt you for a password for the account you specify following the -u argument.
curl -s -k -u jdoe https://sequencing.mbl.edu/downloads/users/jdoe/150207_EDC1.zip > some_other_filename.zip
Downloads a file named 150207_EDC1.zip and saves it into a file named some_other_filename.zip in your current working directory. If you already have a file named some_other_filename.zip in your current working directory, it will be overwritten.
Note: curl will prompt you for a password for the account you specify following the -u argument.
Hint: If you don’t know the exact filename or URL of the file you are trying to download, first open a browser and log into the sequencing facility at https://jbpc.mbl.edu/sequencing/downloads/, once you have authenticated with your username, you will be presented with a list of files available for downloading. Right click on the file you want to download to the remote computer and select the option to copy the “Copy Link or URL”, then you can paste it into the terminal window or a text editor for composing the curl command. (If you don’t have a two button mouse on a Macintosh, click the link while holding down the control key on your keyboard.)
For more information on the terrific and powerful utility ‘curl’, log into a bioware server and type ‘man curl’ without the quotes to get the curl user manual. You can also access this if you are on a Macintosh computer, open the program /Applications/Terminal and type ‘man curl’, again without the quotes.
If you wish to keep your sample plate, find it in the freezer (3rd shelf down from the top). If you don’t want it, it will be discarded as hazardous waste after one week.
If you have questions, please email the sequencing team.